The concerted action of a positive charge and hydrogen bonds dynamically regulates the pKa of the nucleophilic cysteine in the NrdH‐redoxin family

NrdH‐redoxins shuffle electrons from the NADPH pool in the cell to Class Ib ribonucleotide reductases, which in turn provide the precursors for DNA replication and repair. NrdH‐redoxins have a CVQC active site motif and belong to the thioredoxin‐fold protein family. As for other thioredoxin‐fold proteins, the pK_a of the nucleophilic cysteine of NrdH‐redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. Recently, the pK_a value of this cysteine in Corynebacterium glutamicum and Mycobacterium tuberculosis NrdH‐redoxins were determined, but structural insights explaining the relatively low pK_a remained elusive. We subjected C. glutamicum NrdH‐redoxin to an extensive molecular dynamics simulation to expose the factors regulating the pK_a of the nucleophilic cysteine. We found that the nucleophilic cysteine receives three hydrogen bonds from residues within the CVQC active site motif. Additionally, a fourth hydrogen bond with a lysine located N‐terminal of the active site further lowers the cysteine pK_a. However, site‐directed mutagenesis data show that the major contribution to the lowering of the cysteine pK_a comes from the positive charge of the lysine and not from the additional Lys‐Cys hydrogen bond. In 12% of the NrdH‐redoxin family, this lysine is replaced by an arginine that also lowers the cysteine pK_a. All together, the four hydrogen bonds and the electrostatic effect of a lysine or an arginine located N‐terminally of the active site dynamically regulate the pK_a of the nucleophilic cysteine in NrdH‐redoxins.     

Contributions to nucleosome dynamics in chromatin from interactive propagation of phosphorylation/acetylation and inducible histone lysine basicities

The effect of phosphorylation on the basicities of amines in histone H3 peptides and their acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser‐10 lowers the rate of chemical acetylation of Lys‐9, Lys‐14, and Lys‐18 by methyl acetyl phosphate in that order consistent with a higher pK_a of these Lys residues induced by phosphorylation; basicities increase up to 3 pK_a units as a function of distance from Ser‐10 phosphate. Enzymic acetylation of Lys residues with high pK_a values in nucleosomes is also expected to be enhanced by phosphorylation, consistent with the known mechanism involving binding of protonated amines to N‐acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site, its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a phosphate on Ser‐10, the amines of Lys‐9, Lys‐14, and Lys‐18 have lowered pK_a values. Chemical acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but the phosphate inductive effect in histone H3 is more potent since the linkage between phosphorylation and acetylation is propagated with a range extending 9–10 amino acids in either direction from the phosphorylation site enhancing protonation of amino groups. We conclude that lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic and immediate interaction between phosphorylation/acetylation that may contribute to inactive heterochromatin by compaction through such Ser phosphate–Lys amine electrostatic interactions and their relaxation by acetylation in euchromatin.     

Protonation states of active‐site lysines of penicillin‐binding protein 6 from Escherichia coli and the mechanistic implications

The protonation states of the two active‐site lysines (Lys69 and Lys235) of PBP 6 of Escherichia coli were explored to understand the active site chemistry of this enzyme. Each lysine was individually mutated to cysteine, and the resultant two mutant proteins were purified to homogeneity. Each protein was denatured, and its cysteine was chemically modified to produce an S‐aminoethylated cysteine (γ‐thialysine) residue. Following renaturation, the evaluation of the kinetics of the dd ‐carboxypeptidase activity of PBP 6 as a function of pH was found consistent with one lysine in its free‐base (Lys69) and the other in the protonated state (Lys235) for optimal catalysis. The experimental estimates for their pK_a values were compared with the pK_a values calculated computationally, using molecular‐dynamics simulations and a thermodynamic cycle. Study of the γ‐thialysine69 showed that lysine at position 69 influenced the basic limb of catalysis, consistent with the fact that the two lysine side chains are in proximity to each other in the active site. Based on these observations, a reaction sequence for PBP 6 is proposed, wherein protonated Lys235 serves as the electrostatic substrate anchor and Lys69 as the conduit for protons in the course of the acylation and deacylation half‐reactions. Proteins 2014; 82:1348–1358. © 2013 Wiley Periodicals, Inc.     

Potent and Selective Monoamine Oxidase‐B Inhibitory Activity: Fluoro‐ vs. Trifluoromethyl‐4‐hydroxylated Chalcone Derivatives

For various neurodegenerative disorders like Alzheimer's and Parkinson’s diseases, selective and reversible MAO‐B inhibitors have a great therapeutic value. In our previous study, we have shown that a series of methoxylated chalcones with F functional group exhibited high binding affinity toward human monoamine oxidase‐B (hMAO‐B). In continuation of our earlier study and to extend the understanding of the structure–activity relationships, a series of five new chalcones were studied for their inhibition of hMAO. The results demonstrated that these compounds are reversible and selective hMAO‐B inhibitors with a competitive mode of inhibition. The most active compound, (2E)‐1‐(4‐hydroxyphenyl)‐3‐[4‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one, exhibited a K_i value of 0.33 ± 0.01 μm toward hMAO‐B with a selectivity index of 26.36. A molecular docking study revealed that the presence of a H‐bond network in hydroxylated chalcone with the N(5) atom of FAD is crucial for MAO‐B selectivity and potency.     

Origin of the pKa shift of the catalytic lysine in acetoacetate decarboxylase

The pK_a value of Lys115, the catalytic residue in acetoacetate decarboxylate, was calculated using atomic coordinates of the X-ray crystal structure with consideration of the protonation states of all titratable sites in the protein. The calculated pK_a value of Lys115 (pK_a(Lys115)) was unusually low (≈6) in agreement with the experimentally measured value. Although charged residues impact pK_a(Lys115) considerably in the native protein, the significant pK_a(Lys115) downshift in the protein with respect to aqueous solution was mainly due to loss of the solvation energy in the catalytic active site relative to bulk water.     

Bayesian model aggregation for ensemble‐based estimates of protein pKa values

This article investigates an ensemble‐based technique called Bayesian Model Averaging (BMA) to improve the performance of protein amino acid pK_a predictions. Structure‐based pK_a calculations play an important role in the mechanistic interpretation of protein structure and are also used to determine a wide range of protein properties. A diverse set of methods currently exist for pK_a prediction, ranging from empirical statistical models to ab initio quantum mechanical approaches. However, each of these methods are based on a set of conceptual assumptions that can effect a model's accuracy and generalizability for pK_a prediction in complicated biomolecular systems. We use BMA to combine eleven diverse prediction methods that each estimate pK_a values of amino acids in staphylococcal nuclease. These methods are based on work conducted for the pK_a Cooperative and the pK_a measurements are based on experimental work conducted by the García‐Moreno lab. Our cross‐validation study demonstrates that the aggregated estimate obtained from BMA outperforms all individual prediction methods with improvements ranging from 45 to 73% over other method classes. This study also compares BMA's predictive performance to other ensemble‐based techniques and demonstrates that BMA can outperform these approaches with improvements ranging from 27 to 60%. This work illustrates a new possible mechanism for improving the accuracy of pK_a prediction and lays the foundation for future work on aggregate models that balance computational cost with prediction accuracy. Proteins 2014; 82:354–363. © 2013 Wiley Periodicals, Inc.     

A lysine conserved in the monoamine oxidase family is involved in oxidation of the reduced flavin in mouse polyamine oxidase

Lysine 315 of mouse polyamine amine oxidase corresponds to a lysine residue that is conserved in the flavoprotein amine oxidases of the monoamine oxidase structural family. In several structures, this lysine residue forms a hydrogen bond to a water molecule that is hydrogen-bonded to the flavin N(5). Mutation of Lys315 in polyamine oxidase to methionine was previously shown to have no effect on the kinetics of the reductive half-reaction of the enzyme (M. Henderson Pozzi, V. Gawandi, P.F. Fitzpatrick, Biochemistry 48 (2009) 1508-1516). In contrast, the mutation does affect steps in the oxidative half-reaction. The k_cat value is unaffected by the mutation; this kinetic parameter likely reflects product release. At pH 10, the k_cat/K_m value for oxygen is 25-fold lower in the mutant enzyme. The k_cat/K_O2 value is pH-dependent for the wild-type enzyme, decreasing below a pK_a of 7.0, while this kinetic parameter for the mutant enzyme is pH-independent. This is consistent with the neutral form of Lys315 being required for more rapid flavin oxidation. The solvent isotope effect on the k_cat/K_O2 value increases from 1.4 in the wild-type enzyme to 1.9 in the mutant protein, and the solvent inventory changes from linear to bowed. The effects of the mutation can be explained by the lysine orienting the bridging water so that it can accept the proton from the flavin N(5) during flavin oxidation. In the mutant enzyme the lysine amine would be replaced by a water chain.     

TSAR,a new graph–theoretical approach to computational modeling of protein side‐chain flexibility: Modeling of ionization properties of proteins

A new graph–theoretical approach called thermodynamic sampling of amino acid residues (TSAR) has been elaborated to explicitly account for the protein side chain flexibility in modeling conformation‐dependent protein properties. In TSAR, a protein is viewed as a graph whose nodes correspond to structurally independent groups and whose edges connect the interacting groups. Each node has its set of states describing conformation and ionization of the group, and each edge is assigned an array of pairwise interaction potentials between the adjacent groups. By treating the obtained graph as a belief‐network—a well‐established mathematical abstraction—the partition function of each node is found. In the current work we used TSAR to calculate partition functions of the ionized forms of protein residues. A simplified version of a semi‐empirical molecular mechanical scoring function, borrowed from our Lead Finder docking software, was used for energy calculations. The accuracy of the resulting model was validated on a set of 486 experimentally determined pK_a values of protein residues. The average correlation coefficient (R) between calculated and experimental pK_a values was 0.80, ranging from 0.95 (for Tyr) to 0.61 (for Lys). It appeared that the hydrogen bond interactions and the exhaustiveness of side chain sampling made the most significant contribution to the accuracy of pK_a calculations. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Stabilization of C4a-hydroperoxyflavin in a two-component flavin-dependent monooxygenase is achieved through interactions at flavin N5 and C4a atoms

p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C₂), His-396 is 4.5 Å from the flavin C4a locus, whereas Ser-171 is 2.9 Å from the flavin N5 locus. We investigated the roles of these two residues in the stability of the C4a-hydroperoxy-FMN intermediate. The results indicated that the rate constant for C4a-hydroperoxy-FMN formation decreased ∼30-fold in H396N, 100-fold in H396A, and 300-fold in the H396V mutant, compared with the wild-type enzyme. Lesser effects of the mutations were found for the subsequent step of H₂O₂ elimination. Studies on pH dependence showed that the rate constant of H₂O₂ elimination in H396N and H396V increased when pH increased with pK_a >9.6 and >9.7, respectively, similar to the wild-type enzyme (pK_a >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H₂O₂ elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H₂O₂ elimination in S171A and S171T were ∼1400-fold and 8-fold greater than the wild type, respectively. Studies on the pH dependence of S171A with oxygen showed that the rate constant of H₂O₂ elimination increased with pH rise and exhibited an approximate pK_a of 8.0. These results indicated that the interaction of the hydroxyl group side chain of Ser-171 and flavin N5 is required for the stabilization of C4a-hydroperoxy-FMN. The double mutant S171A/H396V reacted with oxygen to directly form the oxidized flavin without stabilizing the C4a-hydroperoxy-FMN intermediate, which confirmed the findings based on the single mutation that His-396 was important for formation and Ser-171 for stabilization of the C4a-hydroperoxy-FMN intermediate in C₂.     

Functional tuning of the catalytic residue pKa in a de novo designed esterase

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p‐nitrophenyl acetate (pNPA) and 4‐nitrophenyl‐(2‐phenyl)‐propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single‐histidine residue (His¹⁴⁴) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK_a value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK_a calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK_a value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK_a value of this residue in water. We determined that a short‐range favorable interaction with Glu¹²⁷ contributes to the elevated pK_a of His¹⁴⁴. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK_a of its active nucleophile, His¹⁴⁴, by 0.7 units. As a direct result of the decrease in the His¹⁴⁴ pK_a value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine‐promoted catalysis. Proteins 2017; 85:1656–1665. © 2017 Wiley Periodicals, Inc.     

The effects of reversible freezing inactivation and inhibitor binding on redox properties of l-amino-acid oxidase

We measured the redox potentials of frozen inactivated l-amino-acid oxidase (l-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) and inhibitor-bound (anthranilic acid) enzyme, and compared these redox properties to those of active l-amino-acid oxidase and benzoate-bound d-amino-acid oxidase (EC 1.4.3.3), respectively. The redox properties of the inactive enzyme are similar to the properties of free flavin; the potential is within 0.015 V of free flavin and no radical stabilization is seen. This corresponds to the loss of most interactions between apoprotein and flavin. In contrast, the anthranilic acid lowers the amount of radical stabilized from 85% to 35%. The potentials are still 0.150 V positive of free flavin, indicating that in the presence of inhibitor, many flavin-protein interactions remain intact. The difference between this behavior and that of d-amino-acid oxidase bound to benzoate, where the amount of radical declined from 95% to 5%, is explained on the basis of the relative tightness of binding of apoprotein to FAD. d-Amino-acid oxidase apoprotein has a relatively low K_a (10⁶) for FAD, and benzoate has a relatively high K_a (10⁵) for the enzyme. Therefore, the binding of benzoate increases the tightness of FAD binding to apo-d-amino-acid oxidase (10¹¹), indicating significant changes in flavin-protein interactions. In contrast, apo-l-amino-acid oxidase binds flavin tightly (the K_a is greater than 10⁷) and the enzyme binds to anthranilate much less tightly, with a K_a of 10³. The l-amino-acid oxidase apoprotein binding to FAD is tight initially, and the binding of anthranilate changes it only slightly. Therefore, redox studies indicate that the ability of a flavoprotein to be regulated may be influenced by the strength of the interaction of flavin with the apoprotein, as well as the strength of interaction of the substrate or activator.     

Enantiomeric resolution of some nonsteroidal antiinflammatory and anticoagulant drugs using β‐cyclodextrins by capillary electrophoresis

β‐cyclodextrin (CD) and its derivatives HP‐β‐CD, DM‐β‐CD, and TM‐β‐CD have been employed as chiral selectors for the separation of three nonsteroidal antiinflammatory drugs (NSAIDs) and anticoagulant at relatively low concentration (8–15 mM) by capillary zone electrophoresis (CZE). In this study, baseline separation was achieved for ibuprofen, ketoprofen, naproxen, and warfarin. It was found that the addition of 0.1% hydroxypropyl methyl cellulose (HPMC) was effective for separation. Under these conditions, the S‐(+) enantiomer eluted before R‐(−) in terms of ibuprofen; the calculated energy values obtained from the molecular modeling correlated well with the elution order. An equation for calculating the pK_a values by capillary electrophoresis was introduced, and the pK_a values of the four chiral drugs at 25°C were obtained based on the equation. The value pK_a + 0.5 is proposed to be the suitable pH of the background electrolyte for the separation of chiral compounds containing a carboxylic group. Chirality 11:56–62, 1999. © 1999 Wiley‐Liss, Inc.     

Binding of FAD and tryptophan to the tryptophan 6‐halogenase Thal is negatively coupled

Flavin‐dependent halogenases require reduced flavin adenine dinucleotide (FADH₂), O₂, and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6‐halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH₂ by flavin reductases.     

Effect of methylation on the side‐chain pKa value of arginine

Arginine methylation is important in biological systems. Recent studies link the deregulation of protein arginine methyltransferases with certain cancers. To assess the impact of methylation on interaction with other biomolecules, the pK_a values of methylated arginine variants were determined using NMR data. The pK_a values of monomethylated, symmetrically dimethylated, and asymmetrically dimethylated arginine are similar to the unmodified arginine (14.2 ± 0.4). Although the pK_a value has not been significantly affected by methylation, consequences of methylation include changes in charge distribution and steric effects, suggesting alternative mechanisms for recognition.     

Purification of glutathione S‐transferase enzyme from quail liver tissue and inhibition effects of (3aR,4S,7R,7aS)‐2‐(4‐((E)‐3‐(aryl)acryloyl)phenyl)‐3a,4,7,7a‐tetrahydro‐1H‐4,7‐methanoisoindole‐1,3(2H)‐dione derivatives on the enzyme activity

The use of quail meat and eggs has made this animal important in recent years, with its low cost and high yields. Glutathione S‐transferases (GST, E.C.2.5.1.18) are an important enzyme family, which play a critical role in detoxification system. In our study, GST was purified from quail liver tissue with 47.88‐fold purification and 12.33% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by SDS‐PAGE method and showed a single band. In addition, inhibition effects of (3aR,4S,7R,7aS)‐2‐(4‐((E)‐3‐(aryl)acryloyl)phenyl)‐3a,4,7,7a‐tetrahydro‐1H‐4,7methanoisoindole‐1,3(2H)‐dion derivatives ( 1a–g ) were investigated on the enzyme activity. The inhibition parameters (IC₅₀ and K_i values) were calculated for these compounds. IC₅₀ values of these derivatives ( 1a–e ) were found as 23.00, 15.75, 115.50, 10.00, and 28.75 μM, respectively. K_i values of these derivatives ( 1a–e ) were calculated in the range of 3.04 ± 0.50 to 131.50 ± 32.50 μM. However, for f and g compounds, the inhibition effects on the enzyme were not found.     

Evidence for mouse sulfhydryl oxidase‐assisted cross‐linking of major seminal vesicle proteins

Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high‐molecular‐weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)‐dependent Sox was purified from mouse SVS using ion exchange and high‐performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The K_m and V_max of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross‐linking activity among SVS I–III monomers to form HMWCs in SVS.     

Differentiation of various types of biological oxidation of nitrogen in organic compounds

The various types of nitrogen which occur in organic compounds and which are susceptible to biological oxidation are clearly divided into groups depending upon the pK_a, of the constituent nitrogen. The enzymatic processes which give rise to the N-oxidation products are reviewed by a consideration of species differences, age of animal, pH optima, influence of inducing agents, inhibitors and microsomal pretreatments, as well as the stereochemistry of the nitrogen atom.From the data collected, a concept is developed which suggests that all basic amines (group I) are oxidised by a flavine adenine nucleotide (FAD)-dependent enzyme system, whereas non-basic nitrogen-containing compounds (group III) are oxidised by a cytochrome P450-dependent system.It is further suggested that compounds of intermediary pK_a,i.e. between 1 and 7 (group II), may be substrates for both enzyme systems, which would yield the same products, but by different processes. The extent to which N-oxidation occurs in a species would therefore depend on the pK_a of the substrate and the amounts and ratio of the two enzymes present, a lower pK_a favouring oxidation by the cytochrome P450 system and a higher pK_a favouring oxidation by the FAD system.In a similar manner, it is suggested that the oxidation of aromatic heterocyclic amines depends upon the pK_a of the nitrogen, compounds having a low pK_a being preferentially metabolised by nitrogen oxidation.     

Combined computational and biochemical study reveals the importance of electrostatic interactions between the “pH sensor” and the cation binding site of the sodium/proton antiporter NhaA of Escherichia coli

Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. The crystal structure of NhaA has provided a basis to explore the mechanism of ion exchange and its unique regulation by pH. Here, the mechanism of the pH activation of the antiporter is investigated through functional and computational studies of several variants with mutations in the ion‐binding site (D163, D164). The most significant difference found computationally between the wild type antiporter and the active site variants, D163E and D164N, are low pK_a values of Glu78 making them insensitive to pH. Although in the variant D163N the pK_a of Glu78 is comparable to the physiological one, this variant cannot demonstrate the long‐range electrostatic effect of Glu78 on the pH‐dependent structural reorganization of trans‐membrane helix X and, hence, is proposed to be inactive. In marked contrast, variant D164E remains sensitive to pH and can be activated by alkaline pH shift. Remarkably, as expected computationally and discovered here biochemically, D164E is viable and active in Na⁺/H⁺ exchange albeit with increased apparent K_M. Our results unravel the unique electrostatic network of NhaA that connect the coupled clusters of the “pH sensor” with the binding site, which is crucial for pH activation of NhaA. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Predicting protein pKa by environment similarity

A statistical method to predict protein pK_a has been developed by using the 3D structure of a protein and a database of 434 experimental protein pK_a values. Each pK_a in the database is associated with a fingerprint that describes the chemical environment around an ionizable residue. A computational tool, MoKaBio, has been developed to identify automatically ionizable residues in a protein, generate fingerprints that describe the chemical environment around such residues, and predict pK_a from the experimental pK_a values in the database by using a similarity metric. The method, which retrieved the pK_a of 429 of the 434 ionizable sites in the database correctly, was crossvalidated by leave‐one‐out and yielded root mean square error (RMSE) = 0.95, a result that is superior to that obtained by using the Null Model (RMSE 1.07) and other well‐established protein pK_a prediction tools. This novel approach is suitable to rationalize protein pK_a by comparing the region around the ionizable site with similar regions whose ionizable site pK_a is known. The pK_a of residues that have a unique environment not represented in the training set cannot be predicted accurately, however, the method offers the advantage of being trainable to increase its predictive power. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pKa values

The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65‐residue‐long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pK_a values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C‐terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pK_a values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random‐coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N‐terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7.